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Conventional reporter gene technology and histological methods cannot routinely be used to track the in vivo behavior of embryonic stem (ES) cells longitudinally after cellular transplantation. Here we describe a protocol for monitoring the in vivo survival, proliferation, and migration of ES cells following surgical administration without necessitating animal sacrifice. Stable ES cell lines containing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes can be established within 4–6 weeks by lentiviral transduction followed fluorescence activated cell sorting (FACS). The cell fate and behavior of these DF or TF ES cells can subsequently be tracked non-invasively by bioluminescence and microPET imaging for a prolonged period of time.
Embryonic stem (ES) cells offer exciting promises as therapeutic donor cells for regenerative medicine. Unlike adult stem cells, ES cells can differentiate into any somatic cell of the human body and have the capacity for unlimited self renewal 1. To fully realize the therapeutic potential of ES cells, however, it is important for investigators to understand the in vivo behavior of transplanted cells following administration. At present, a number of hurdles have hindered the effective translation of ES-cell based therapy to the clinic. These issues include teratoma formation, immune rejection, failure of cells to engraft, and cellular migration outside the area of administration. The development of sensitive and accurate methods to track the survival, proliferation, and migration of ES cells or ES cell derivatives will thus be indispensable for future applications of ES cell therapy in human patients.
In this chapter, we describe a novel method of monitoring in vivo ES cell behavior through the use of bioluminescence imaging (BLI) and positron emission tomography (PET) reporter genes. In these types of cellular imaging, a reporter gene coding for the synthesis of a detectable protein is stably introduced into the genome of a target cell or tissue via a lentiviral vector carrying a constitutively active promoter such as ubiquitin that drives reporter gene expression. Subsequent cell mediated synthesis of the reporter protein produces a probe that can interact with an exogenously administered substrate to generate a detectable signal (Figure 1). In the case of BLI, the reporter gene introduced is firefly luciferase (Fluc). Interaction of Fluc with its substrate D-luciferin catalyzes production of the optically active metabolite oxyluciferin which emits low intensity photons that can be imaged with a cool-charged couple device (CCD) camera for cell localization. In PET imaging, the reporter protein herpes simplex virus thymidine kinase (HSVtk) phosphorylates its substrate, the PET reporter probe 9-4-[18F]fluoro-3-(hydroxylmethylbutyl) guanine ([18F]-FHBG), to produce high-energy photons that can be captured by a PET camera. Our group has used both a double fusion (DF) construct containing enhanced green fluorescent protein (eGFP) and Fluc reporter genes, and a triple fusion (TF) construct containing monomeric red fluorescent protein (mRFP), Fluc, and HSVtk reporter genes, to stably transduce ES cells for reporter gene imaging
As compared to conventional methods of assaying cell fate such as histological analysis and staining for GFP or β-galactosidase (LacZ), reporter gene imaging allows for non-invasive and longitudinal visualization of the spatio-temporal kinetics of cell engraftment and survival in living subjects without requiring animal sacrifice. Because cellular transcription and translation must be intact for synthesis of the reporter proteins, only cells that are alive generate positive signal. In addition, genetic inheritance of the reporter gene from mother to daughter cell permits longitudinal monitoring of cellular proliferation and misbehavior (e.g., teratoma formation). Previous studies have also shown that expression of the reporter genes do not significantly impact ES cell viability, proliferation, and differentiation 2–4. Our laboratory has applied reporter gene imaging to successfully monitor the in vivo survival, migration, and proliferation of transplanted ES cells 4–7 and ES cell derivatives such as cardiomyocytes 8 and endothelial cells 9, 10 over a prolonged period of time.
The following steps describe the derivation of murine and human ES cell lines that stably express DF (eGFP-Fluc) and TF (mRFP-Fluc-HSVtk) reporter genes, as well as imaging protocols to monitor the survival, migration, and proliferation of these cells. Because the emphasis of this chapter is on reporter gene transduction and imaging, we have not included instructions on how to make the reporter gene plasmid constructs used in Section 3.1 below. For a description of the PCR and standard molecular cloning techniques used to make these constructs please refer to De et. al 11, Ray et. al 12, and Cao et. al 4.
This work was supported by Howard Hughes Medical Institute research fellowship (AL), R21 HL091453 (JCW), and R21/R33 HL089027 (JCW).